論文

生理的範囲での[K+]oの変化がアストロサイトのエネルギー基質利用率に与える影響を検討した.更に高グルコース(22mM)DMEMと低グルコース(2mM)DMEMで培養したアストログリアの反応の差異についても検討した.尚,アストログリアは新生仔Sprague-Dawleyラット大脳皮質より組織を採取し培養を行った.1)グルコース2mM下で培養した細胞では,22mMに比べ[14C]lactate酸化率の上昇を認めた.[14C]deoxyglucoseリン酸化率は変化しなかった.2)細胞外K+濃度の上昇により,培養アストログリアの[14C]deoxyglucoseリン酸化率は増加し,[14C]lactate 酸化率は変化しなかった.3)神経活動亢進時に放出されるK+を介し,アストロサイトの解糖系エネルギー消費のみが亢進する可能性が示唆された

38歳男.頸部整体治療中に突然,右片麻痺,深部感覚障害および左舌下神経麻痺を発症した.脳血管撮影およびMR angiographyにて左椎骨動脈の狭窄,不整拡張などが認められた.MRI上,延髄左内側部に梗塞巣が確認され,延髄内側梗塞と診断された.発症機序としては,頸部整体術による頸部回旋運動で椎骨動脈に過度に伸展,圧排が加わることにより血管内膜損傷を生じ,動脈解離を引き起こすことにより虚血に陥り,延髄梗塞を発症したと考えられた.臨床経過は全般的に改善傾向がみられ約2ヵ月半後退院となったが,右上肢麻痺の改善は認められなかった.本症の発症を少しでも未然に防ぐには,整体術を受ける前に頭頸部を静かに回旋,過伸展させた状態でいったん経過観察し,何らかの異常を訴える場合は即時中止すべきである

Recently, it has been hypothesized that neurons mainly utilize lactate produced by astrocytes to generate energy (i.e. ATP). Regardless of numerous studies, whether lactate rather than glucose fuels neurons in vivo remains a matter of controversy. In the present study we have examined the substrate-preference of the reduction of Alamar Blue, a novel redox indicator, by cultured rat astroglia and neurons. Alamar Blue reduction is dependent on cellular reducing equivalents. Therefore, the substrate-preference of Alamar Blue reduction could reflect that for cellular energy production. Rates of Alamar Blue reduction by 2 mM glucose were two-fold higher in astroglia than those in neurons. Alamar Blue reduction induced by 2 mM lactate was 78% of that by 2 mM glucose in neurons while only 20% in astroglia. Nevertheless, glucose elicited larger Alamar Blue reduction than lactate both in astroglia and neurons. These results indicate that neuronal energy production in vivo depends on both the glycolytic and oxidative metabolism of glucose even though astroglia rely on glycolysis as has been reported.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is widely used to evaluate cell proliferation and viability. MTT reduction is interpreted to be indicative of cellular metabolic activity, and the site of reduction includes both mitochondrial and cytosolic redox reactions. Astrocytes are believed to rely mainly on glycolysis for ATP generation, whereas neurons are considered to depend more on oxidative metabolism. The present study, therefore, tested the substrate-preference of glucose and its metabolites for MTT reduction in cultures of rat type 1 astroglia and neurons.MTT specific activity of astroglia was much higher than that of neurons. Astroglial MTT reducing activity in glucose-free medium or 2mM glucose with iodoacetate (5mM) was completely blocked. In glucose-depleted medium, 2mM lactate, pyruvate, malate, or acetate elicited minimal increases in MTT reduction by astroglia. In contrast, MTT reducing activity in neurons was enhanced two-fold by pyruvate and the reducing activity of lactate was equivalent to that of glucose, while malate had a small and acetate had no effect on MTT reduction. These results indicate that these two cell types differ markedly in their substrate-preferences for MTT reduction. In astroglia, MTT reduction reflects mainly cytosolic redox activity and is dependent on glyceraldehyde-3-phosphate dehydrogenase. In neurons, pyruvate dehydrogenase supports MTT reduction more effectively than glucose or lactate, even though both of these substrates can produce NADH and pyruvate.

Na(+) influx has been implicated to play an important role in the mechanisms of neuronal cell damage under ischemia as well as in neurodegenerative disorders. Thus far, however, the effects of Na(+) influx on astrocytic damage have not been studied extensively. In the present study, we have examined the effects of Na(+) influx induced by veratridine (Na(+) channel opener), monensin (Na(+) ionophore), and glutamate (co-transportation with Na(+)) on rat cultured astroglial damage. Cells were incubated with bicarbonate buffer with 25 mM glucose containing either 100 microM veratridine, 10 microM monensin, or 1 mM glutamate with or without 1 mM ouabain for 20 h. Cellular damage was evaluated quantitatively by lactate dehydrogenase (LDH) release or 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction. Veratridine, monensin, or glutamate alone did not induce significant astroglial damage. Veratridine and monensin co-incubated with ouabain, which inhibits active extrusion of Na(+) by Na(+),K(+)-ATPase, thereby enhances intracellular Na(+) accumulation, caused significant cell death (P<0. 001, approximately 50% cell damage), whereas glutamate did not. Na(+)-free solution substituted by choline (impermeable cation) attenuated cell damage induced by veratridine and monensin markedly, while Li(+) substitution (permeable cation) rather exacerbated. Nifedipine (100 microM), a blocker of L-type Ca(2+) channel, reduced veratridine-induced glial damage by 50%. Neither bepridil nor benzamil, a blocker of Na(+)-Ca(2+) exchanger, had any protection. Cyclosporin A (1 or 10 microM), an inhibitor of mitochondrial permeability transition or 10 microM N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (zVAD-fmk), which inhibits a broad range of caspases, did not show protective effects.

MTT還元反応は,アストロサイトでは解糖系,ニューロンではTCAサイクルを中心としたエネルギー産生能と,これによるredox stateを反映し,必ずしもミトコンドリア機能のみの指標ではない

Cerebral blood flow (CBF) rises when the glucose supply to the brain is limited by hypoglycemia or glucose metabolism is inhibited by pharmacological doses of 2-deoxyglucose (DG). The present studies in unanesthetized rats with insulin-induced hypoglycemia show that the increases in CBF, measured with the [14C]iodoantipyrine method, are relatively small until arterial plasma glucose levels fall to 2.5 to 3.0 mM, at which point CBF rises sharply. A direct effect of insulin on CBF was excluded; insulin administered under euglycemic conditions maintained by glucose injections had no effects on CBF. Insulin administration raised plasma lactate levels and decreased plasma K+ and HCO3- concentrations and arterial pH. These could not, however, be related to the increased CBF because insulin under euglycemic conditions had similar effects without affecting CBF; furthermore, the inhibition of brain glucose metabolism with pharmacological doses (200 mg/kg intravenously) of DG increased CBF, just like insulin hypoglycemia, without altering plasma lactate and K+ levels and arterial blood gas tensions and pH. Nitric oxide also does not appear to mediate the increases in CBF. Chronic blockade of nitric oxide synthase activity by twice daily i.p. injections of NG-nitro-L-arginine methyl ester for 4 days or acutely by a single i.v. injection raised arterial blood pressure and lowered CBF in normoglycemic, hypoglycemic, and DG-treated rats but did not significantly reduce the increases in CBF due to insulin-induced hypoglycemia (arterial plasma glucose levels, 2.5-3 mM) or pharmacological doses of deoxyglucose.

Studies of local glucose utilization in neural tissues in vivo with the autoradiographic [14C]deoxyglucose method have demonstrated that energy metabolism increases almost linearly with the degree of functional activation, i.e. spike frequency, in the terminal projection zones of activated pathways. The increased metabolism is found in neuropil and is minimal or undetectable in neuronal cell bodies. Electrical stimulation, increased extracellular [K+] ([K+]o), or opening of Na+ channels with veratridine stimulates metabolism in neutral tissues, and this increase is blocked by ouabain, a specific inhibitor of Na+,K(+)-ATPase. Activation of this enzyme to restore ionic gradients across cellular membranes appears to mediate the function-related increase in energy metabolism. The metabolic activation is, therefore, not directly related to the functional activity itself but to processes operating to recover from that activity. The limited spatial resolution of the [14C]DG method precludes identification of cellular elements in neuropil participating in the metabolic activation, e.g. axonal terminals, dendrites, or astrocytic processes enveloping the synapses. We have, therefore, attempted to stimulate in vitro conditions to be expected from functional activation and increased spike activity in vivo, e.g. increased extracellular [K+], intracellular [Na+], or extracellular neurotransmitter levels, and examined their effects on glucose metabolism in neurons and astroglia in culture. Increased [K+]o stimulated [14C]DG phosphorylation in neuronal and mixed neuronal-astroglial cultures, but not in astroglial cultures assayed in bicarbonate buffer; it did occasionally stimulate metabolism in astroglia when assayed in HEPES or phosphate buffers, but these effects were variable and inconsistent. Veratridine (75 microM) stimulated [14C]DG phosphorylation in neurons and astroglia; these stimulations were blocked by 1 mM ouabain or 10 microM tetrodotoxin (TTX), which blocks voltage-dependent Na+ channels. The Na+ ionophore monensin (10 microM) doubled the rate of metabolism, a stimulation that was only partially blocked by ouabain and unaffected by TTX. L-Glutamate (500 microM) stimulated [14C]DG phosphorylation in astroglia, but this stimulation was probably secondary to Na+ uptake into the cells via a sodium/glutamate co-transporter because it was not blocked by inhibitors of NMDA or non-NMDA receptors but was absent in Na(+)-free medium. These results indicate that astroglia contribute to the increased energy metabolism in neuropil during functional activation by mechanisms that promote Na+ entry into the cells.

The effects of chronic treatment with NG-nitro-L-arginine methyl ester, a potent inhibitor of nitric oxide synthase activity, on local cerebral glucose utilization were examined in conscious rats. Intraperitoneal injections of 50 mg/kg of the nitroarginine twice daily for 4 days have been found to result in almost complete inhibition of nitric oxide synthase activity in brain. Local cerebral glucose utilization was determined by means of the quantitative autoradiographic [14C]deoxyglucose method in an experimental group (n = 7) of rats that were treated with the nitroarginine according to this schedule and in a normal control group (n = 7) treated similarly with saline. The rats were conscious but partially restrained during the determinations of local cerebral glucose utilization. The nitroarginine treatment raised mean arterial blood pressure statistically significantly to 147 +/- 3 mm Hg (mean +/- SEM) from a level of 120 +/- 5 mm Hg in the saline controls (p < 0.001 by grouped t test), but there were no statistically significant effects on glucose utilization in any of 39 brain structures examined. It is concluded that nitric oxide normally exerts no significant influence on energy metabolism in the rat brain.

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.